The long-term goal of this research is to understand how central neuron synapses form. Our current focus is to test the function of neurexins in synapse formation between hippocampal neurons. Beta-neurexins bind to neuroligins-1, a component of glutamate postsynaptic sites. Since neurexins bind synaptotagmin and presynaptic PDZ proteins including CASK and Mints, and neuroligins bind postsynaptic PDZ proteins including PSD-95, it has been suggested that the trans-synaptic neurexin-neuroligin interaction is involved in initiation of glutamatergic synapses. Furthermore, neuroligins expressed on the surface of fibroblasts can induce clustering of synaptic vesicles in contacting axons, both glutamate and GABA axons. In preliminary experiments, we found that neurexin expressed on the surface of fibroblasts induces clustering of the excitatory postsynaptic scaffolding protein PSD-95 and NMDA glutamate receptors, and of the inhibitory postsynaptic scaffolding protein gephyrin and GABA receptors, in contacting dendrites. Other proteins distantly related to neurexins including agrin cannot cluster either glutamate or GABA postsynaptic components. A putative role for neurexins at GABA synapses is supported by the expression and presynaptic localization of neurexins in GABA as well as glutamate axons. Thus the present state of knowledge suggests a central role for neurexins in both glutamate and GABA synapse formation, but our knowledge is incomplete. We propose to test the hypothesis that different isoforms of neurexins, and perhaps neuroligins, promote differentiation of glutamate and GABA synapses. We will define the residues of neurexin-lb necessary for inducing clustering of GABA and glutamate postsynaptic components, and test the possibility that different neuroligin isoforms mediate these effects. We will test the other neurexin isoforms la, 2a/b, and 3a/b for similar inducing and binding activity. We will determine the subcellular distributions of different neuroligin isoforms, and test their ability to induce presynaptic differentiation in GABA and glutamate axons. Finally, we will use two approaches to knock out the function of neurexins in hippocampal cultures and determine whether neurexins are essential for any aspect of synaptogenesis. These experiments address fundamental mechanisms of brain development with implications for therapeutic approaches to regeneration following neurological disorders or trauma.